Gonorrhea remains a global public health problem with over 106 million cases estimated by WHO in 2008 and over 334,000 cases reported to the US CDC in 2012, and rates are on the rise. With increasing Neisseria gonorrhoeae (GC) antibiotic resistance and no new antimicrobial therapies in the pipeline, the possibility of incurable gonorrhea is uncomfortably real. The potential adverse health consequences of untreatable GC and attendant increases in HIV transmission are alarming and highlight the urgent need for new therapeutic and vaccine targets. Our long term goals are to understand the molecular mechanisms of GC pathogenesis and identify bacterial structures required for infection with the aim of discovering potential novel targets for GC vaccines and/or treatments. In nature, GC only infects humans, which limits the extent to which results in animal models are clinically translatable. Accordingly, we propose a clinical trial with the immediate goals of 1) determining the requirement for several GC cell envelope structures in male urethral infection and 2) characterizing human immune responses to mutants lacking these structures. The expected outcome of the trial is the identification of one or more GC envelope structures required for human infection, which will validate these structures as potential novel targets for GC vaccines or treatments. The assembled research team manages the experimental human gonococcal infection program at the University of North Carolina, which is currently the only one of its kind in the world, providing a unique opportunity to study GC factors and host responses in human infection under controlled conditions. Experimental urethral infection of male volunteers is safe, and wild-type GC elicit signs and symptoms of natural gonorrhea. The proposed trial focuses on several surface structures that mediate GC resistance to human innate immune responses. We hypothesize that experimental male urethral infection with GC strains containing mutations that alter or eliminate these structures will show reduced mutant infectivity, decreased inflammation or increased GC mutant clearance compared to wild-type GC. We will use noncompetitive infections to measure GC survival and host immune responses in addition to changes in GC gene expression. We propose studies of three GC envelope structures that we have shown mediate resistance to human innate immune cells and human-derived antimicrobial compounds in the lab and in a mouse model. Aim 1 focuses on mutations affecting the GC MtrCDE efflux pump, which exports host antimicrobial compounds and is important for GC infection in human cell systems and in murine genital infection. Aim 2 focuses on mutations that alter the structure of lipid A, which is important for intracellular GC survival in vitro, and influences GC survival in mice. Finally, Aim 3 focuses on mutations in GC enzymes that cause release of peptidoglycan subunits that modulate innate immune responses in human cell systems and in mice. The GC envelope structures that we aim to test in the trial have not been well-studied in human infection, and they represent novel potential therapeutic and/or vaccine targets.